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Myeloproliferative neoplasms (MPNs) are caused by somatic pathogenic variants that stimulate increased production and clonal expansion of CD34 multipotent hematopoietic stem cells. Recent World Health Organization (WHO) diagnostic criteria for the diagnosis of Philadelphia chromosome (Ph) negative MPNs includes detection of mutations in the Janus Kinase 2 (), myeloproliferative leukemia (), and calreticulin () genes. The purpose of this study was to demonstrate the clinical utility of an in-house next-generation sequencing (NGS) assay targeting only these canonical genes for the molecular diagnosis of patients with Ph-negative MPNs. We tested 274 samples of patients clinically suspected of having Ph-negative MPNs using an in-house developed NGS panel. The assay consists of two parts, a multiplexed PCR and a highly multiplexed NGS workflow capable of handling diverse samples. The assay is capable of simultaneously detecting mutations in exons 12 and 14, exon 9, and exon 10. Of the 274 samples tested, 49 samples harbored mutations in the gene (48 for the V617F and 1 for exon 12), 31 harbored mutations in the gene, and two harbored mutations in the gene. One sample harbored a mutation each in the and genes. Here, we present the distribution of mutations in an Indian cohort of 274 patients from India with Ph-negative MPNs. Moreover, we have successfully demonstrated the clinical utility of our in-house multiplexed NGS assay for the molecular diagnosis of MPNs with varying mutation depths

Aim: The aim of this study is to evaluate the effect of different bonding techniques ethanol wet bonding and dimethyl sulfoxide (DMSO) wet bonding and a novel collagen cross-linker Quercetin application on the durability of resin-dentin bond and observe the bonded interface under the scanning electron microscope (SEM). Materials and Methods: For shear bond strength testing, flat coronal dentin surfaces were prepared on 110 extracted human molars. Teeth were randomly divided into five experimental groups according to different surface pretreatments techniques. Group A was control group without any surface pretreatment. In Group B, ethanol wet bonding pretreatment was done before the application of adhesive. In Group C, DMSO wet bonding was done before the application of adhesive and in Groups D and E, Quercetin along with ethanol and Quercetin along with DMSO pretreatment, respectively, were done before adhesive application. Composite restorations were placed in all the samples. Twenty samples from each group were subjected to immediate and delayed (9 months) shear bond strength evaluation. In addition, two samples per group were subjected to the scanning electron microscopic analysis for the observation of resin-dentin interface. Statistical Analysis: Data collected were subjected to the statistical analysis using the one-way analysis of variance and post hoc Tukey's test at a significance level of P < 0.05. Results: Dentin pretreatment with all the techniques resulted in significantly higher resin-dentin bond strength after 9 months storage with DMSO group showing the highest bond strength values. Conclusion: It can be concluded that these biomodification techniques can improve the durability of the resin-dentin bond.

Atypical haemolytic uremic syndrome (aHUS) is a clinically and genetically heterogeneous condition caused by a complex interplay between genomic susceptibility factors and environmental influences. Pathogenic variants in the DGKE gene are recently identified in cases with infantile-onset autosomal recessive aHUS. The presence of low serum C3 levels, however, has rarely been described in cases of DGKE-associated aHUS. Molecular genetic testing was performed by a commercial next-generation sequencing (NGS) panel as well and by an in-house developed targeted NGS for DGKE gene. Copy number variations (CNVs) were computed from NGS data by calculating a normalised copy number ratio of aligned number of reads at targeted genomic regions against multiple reference regions of the same sample and multiple controls. We report here two such novel clinically relevant variants (c.727_730delTTGT and c.251_259delGCGCCTTC) in the DGKE gene, in two families of infantile aHUS with low serum C3 levels.

Dubin-Johnson syndrome (DJS), an autosomal recessive disorder presenting with conjugated hyperbilirubinemia, is not associated with progression to chronic liver disease (CLD). Next-generation sequencing, application of bioinformatics pipeline, and segregation analysis were performed on 8 members of a consanguineous family with DJS and CLD. A novel variant, c.4406_4407delTA (p.Leu1469fs), in the ABCC2-gene in a homozygous state was found to be associated with DJS and CLD in proband and afflicted family members. DJS may not be a benign entity and novel genetic variants may be associated with progressive liver disease.

Beta thalassemia major is a common genetic disorder characterized by the reduced production or absence of beta globin, a product of the haemoglobin subunit beta (HBB) gene. Every year, approximately 10,000–12,000 children with thalassemia major are born in India. Molecular methodologies like ARMS (amplification-refractory mutation system)-PCR (polymerase chain reaction) and capillary sequencing are used to detect HBB gene mutations. It is, however, challenging to achieve comprehensive coverage of the HBB gene by these methods. Next-generation sequencing (NGS) can be used to circumvent these problems. Commercial NGS panels are prohibitively expensive and hence are not routinely implemented in most laboratories. We have developed a cost-effective, highly sensitive and specific, indigenous targeted NGS assay for detecting mutations in the HBB gene. Using this custom NGS assay, we processed 1530 samples (3017 alleles), in which we detected a spectrum of 48 pathogenic/likely pathogenic variants (mutations); IVS-I-5 (c.92+5G>C) was the most common mutation detected (allele frequency (AF) 44.55%), followed by the 619-bp deletion (AF 10.74%), c.92+1G>T (AF 6.99%), c.27_28insG (AF 6.23%), c.47G>A (AF 5.77%) and c.126_129delCTTT (AF 4.71%). Additionally, we discovered a novel mutation (c.7delC) that was submitted to the HbVar database (HbVar ID 3193) as a variant of unknown significance (VUS), probably pathogenic. The targeted NGS assay developed during this study was validated using orthogonal methods and showed excellent correlation with currently available molecular methods. Additionally, this targeted NGS assay was used to analyse the mutation spectrum of the largest beta thalassemia cohort from India.

Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband’s and parent’s(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

Background Mycobacterium tuberculosis (Mtb) drug resistance is a global concern. Moreover, multiple drug resistant (MDR), extensively drug resistant (XDR), and totally drug resistant (TDR) Mtb cases are on the rise in developing countries like India. Most of these cases are identified only 3–6 months after initiation of treatment owing to incomplete/failed clinical response and incomplete information from phenotypic drug resistance assays and/or targeted Mtb mutation analysis. Here, we report the development of an in-house whole genome sequencing (WGS) assay and bioinformatics pipeline that helped resolve the phenotype-genotype discrepancy in a clinical isolate. Methodology and results A sample from a suspected drug resistant Mtb case tested by line probe assay (LPA) showed the absence of both the mutant and wild type alleles for an rpoB gene mutation site. An in-house next generation sequencing (NGS) assay was used for WGS of this isolate. Bioinformatics analysis revealed that the isolate harboured a novel insertional mutation in the 81-bp hotspot region of the rpoB gene and a S315T mutation in the katG gene, which could explain resistance to rifampicin and isoniazid, respectively. These results correlated with the clinical diagnosis, LPA, solid culture drug susceptibility testing, and pyrosequencing carried out on the sample. The WGS data also provided information regarding the isolate’s lineage and indicated an absence of known mutations conferring resistance to other antitubercular drugs. Conclusion WGS is a highly sensitive, specific, and unbiased approach for identification of all possible drug resistance-conferring mutations, which can help clinicians make more informed treatment-related decisions.

Spinal muscular atrophy (SMA) is one of the common autosomal recessive disorders with global heterozygous carrier frequency of 1:50. Due to high carrier frequency, significant morbidity associated with the infantile onset disease and prohibitive cost of recently approved therapy, American College of Medical Genetics and Genomics (ACMG) recommends population based screening for SMA carrier status in eligible individuals in the reproductive age group. CODE-SEQ is a novel proprietary next generation sequencing (NGS) based assay, which is capable of detecting homozygous as well as heterozygous SMN1 exon 7 deletions. Along with the copy number estimation, this assay is capable of detecting single nucleotide polymorphisms (SNPs) associated with silent SMA carrier status or “2+0” genotype. We have validated a proprietary CODE-SEQ technology in a blinded cohort of 80 clinically well characterized samples from Turkish population for the detection of SMA carriers as well as affected cases. The results were correlated with gold standard MLPA assay. The copy numbers in exon 7 of SMN1 gene matched with MLPA results in all 80 samples giving 100% correlation. The assay accurately detected the presence/ absence of SNPs associated with “2+0” genotype in the reference samples. None of the tested clinical samples had these SNPs. The results of this study support the notion that CODE-SEQ will be extremely useful in detecting SMA genotypes in large-scale population-based screening studies.

BACKGROUND AND OBJECTIVES Pregnancy-associated hemolytic uremic syndrome (P-aHUS) is an important cause of peripartum acute kidney injury). Studies from Europe have described mutations in complement regulator genes, while the data in Indian P-aHUS patients is scarce. METHODS We present 17 patients of P-aHUS who were investigated for complement protein levels and genetic analysis with multiplex ligation-dependent probe amplification (MLPA) for complement genes. Plasma exchange therapy was offered to all patients presenting in acute phase. RESULTS Mean age 26.74 (3.36) years with 15/17 delivered by cesarean section. Eleven patients received early (within 7 days) plasma exchange, three were dialysis-dependent at 3 months and seven were dialysis-free. Only one of the three patients receiving late (after 7 days) plasma exchange was dialysis-free. MLPA showed that 11 patients had heterozygous deletions of exons 3, 5, 6 of CFHR1 and upstream region of exons 1, 2, 3, 6 and intron 4 of CFHR3 gene while four patients had homozygous deletions at the same loci. Two patients had no MLPA-detectable variations. CONCLUSIONS This study reports a high proportion of deletions of exons of CFHR1 & CFHR3 genes in Indian P-aHUS patients detectable by MLPA by copy number variations. This needs confirmation in large multi-center studies. Plasma exchange can be an effective therapy in the non-availability of Eculizumab. This article is protected by copyright. All rights reserved.

Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of clonal disorders that are characterized by excessive proliferation of abnormal myeloid precursors and mature cells. Somatic driver mutations in the JAK2, CALR, and MPL genes serve as major diagnostic criteria in the classification of the MPNs, namely polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Although initially thought to be mutually exclusive, recent studies have reported the co-existence of JAK2, MPL, and CALR mutations. In this case report, we describe a case of a Philadelphia-negative myeloproliferative neoplasm harboring mutations in the CALR [NM_004343.3:c.1092_1143del52 (NP_004334.1:p.Leu367Thrfs)] and MPL [NM_005373.2:c.1543T>A (NP_005364.1:p.Try515Arg)] genes. Given the rarity of documented co-occurrence of driver mutations in these two genes and the concomitant paucity of data regarding management of patients harboring mutations in both these genes simultaneously, there are no clear guidelines for the treatment of patients with these mutation patterns and hence it is difficult to assess the true relevance of this genotype. The fact that both the MPL and CALR genes are a part of the JAK-STAT pathway could, however, assist in the clinical decision-making process.

Introduction and Aims: Dexmedetomidine, an ?-2-adrenoreceptor agonist, offers excellent sedation without significant effect on respiratory or cardiovascular stability. It may be, thus, helpful to achieve smooth extubation. This study was conducted to assess the efficacy of dexmedetomidine in attenuating hemodynamic responses and airway reflexes encountered during extubation. Materials and Methods: Sixty ASA I & II patients, aged 18-50 years, undergoing elective surgery under general endotracheal anesthesia were enrolled for the study. They were assigned to two groups-1 and 2, of 30 patients each, in a randomized, double blind manner, to receive dexmedetomidine or normal saline (NS) intravenous infusion, respectively. Hemodynamic parameters (heart rate, systolic, diastolic and mean arterial pressures) were recorded at the time of starting the study drug infusion, during the infusion, at extubation and thereafter at specific intervals for 30 minutes. Quality of extubation was assessed on a 5-point scale; postoperative sedation assessed by Ramsay Sedation Scale. Any untoward occurrence like undue sedation, vomiting, hypotension, respiratory depression, laryngospasm, bronchospasm and desaturation was noted. Results: Heart rate, systolic, diastolic and mean arterial pressures were significantly higher in Group 2 (p Conclusion: Dexmedetomidine 0.5 mcg/kg infusion given over 10 minutes prior to extubation, decreases hemodynamic responses and results in smooth extubation. Keywords : ?-2-adrenoreceptor agonist, Dexmedetomidine, General anesthesia, Extubation, Hemodynamics.

Familial hypercholesterolaemia (FH) is associated with severely elevated serum low-density lipoprotein cholesterol (LDL-C) levels that lead to premature onset coronary artery disease (CAD) and is r...

ContextMutations in genes encoding the lipoprotein lipase enzyme, its cofactor, or transport proteins can cause severe familial hypertriglyceridemia, resulting in serious complications, such as severe pancreatitis, hepatosplenomegaly, lipid encephalopathy, and failure to thrive. Current treatment includes a low-saturated-fat formula enriched with high medium-chain triglyceride (TGs), oral fibrates, omega-3 fatty acids, or plasmapheresis.Case DescriptionA 71-day-old infant with very severe hypertriglyceridemia and recurrent pancreatitis associated with a likely pathogenic variant in the LPL gene was treated successfully with insulin infusion and a locally prepared low-fat formula feed after stopping breast milk. Subcutaneous insulin was administered daily from 9 to 30 months of age. His serum TG level was markedly lower, although higher than normal. No episodes of hypoglycemia were noted. Fenofibrate and omega-3 fatty acids were ineffective in this infant. At the last follow-up visit, he was 36 months old and growing normally. He was consuming a special meal plan and receiving insulin injections during high-fat meals. Two other young infants with severe hypertriglyceridemia were growing normally after a short course of insulin infusion and the same modified reduced long chain fat diet.ConclusionsInsulin is an unusual and affordable therapeutic option for some patients with severe hypertriglyceridemia and can be helpful in the prevention of acute and chronic complications. Locally available cereals and millets with high crude fiber and a low glycemic index, along with medium chain TGs, was used to prepare an economical special formula at home to maintain TG concentrations in the acceptable limits.

Background & objectives: Prenatal diagnosis of malformations is an important method of prevention and control of congenital anomalies with poor prognosis. Central nervous system (CNS) malformations amongst these are the most common. The information about the prevalence and spectrum of prenatally detected malformations is crucial for genetic counselling and policymaking for population-based preventive programmes. The objective of this study was to study the spectrum of prenatally detected CNS malformations and their association with chromosomal abnormalities and autopsy findings. Methods: This retrospective study was conducted in a tertiary care hospital in north India from January 2007 to December 2013. The details of cases with prenatally detected CNS malformations were collected and were related with the foetal chromosomal analysis and autopsy findings. Results: Amongst 6044 prenatal ultrasonographic examinations performed; 768 (12.7%) had structural malformations and 243 (31.6%) had CNS malformations. Neural tube defects (NTDs) accounted for 52.3 per cent of CNS malformations and 16.5 per cent of all malformations. The other major groups of prenatally detected CNS malformations were ventriculomegaly and midline anomalies. Chromosomal abnormalities were detected in 8.2 per cent of the 73 cases studied. Foetal autopsy findings were available for 48 foetuses. Foetal autopsy identified additional findings in eight foetuses and the aetiological diagnosis changed in two of them (4.2%). Interpretation & conclusions: Amongst prenatally detected malformations, CNS malformations were common. NTD, which largely is a preventable anomaly, continued to be the most common group. Moreover, 60 per cent of malformations were diagnosed after 20 weeks, posing legal issues. Chromosomal analysis and foetal autopsy are essential for genetic counselling based on aetiological diagnosis.

To screen for variants in the MC4R and LEP genes in 46 patients with clinical suspicion of non-syndromic early onset severe obesity (NEOSO). Children with early onset obesity satisfying WHO criteria of obesity were studied. The MC4R and LEP genes were sequenced using a PCR amplicon based NGS on Illumina MiSeq next generation sequencer using an in-house developed protocol. Of the 46 children tested, four were found to have novel pathogenic/likely-pathogenic variants (one in the MC4R gene and three in the LEP gene). In three out of the 4 families, the presence of the variants was confirmed using standard bidirectional capillary sequencing in the probands. Four children with novel likely pathogenic variants in the MC4R and LEP genes are reported. Genetic analysis is crucial in children with early onset obesity and should be considered.

Familial cirrhosis is a condition that is associated with the presence of liver disease with genetic linkage among multiple family members in a generation or in multiple generations. With cirrhosis, most of these disease pathogeneses are related to a defect of an enzyme/transport protein leading to a deranged metabolic pathway with variable prevalence. Many studies and high-quality metanalyses have shed light on genetic linkage associated with nonalcoholic fatty liver disease and steatohepatitis such as the PNPLA3, MBOAT7, and TM6SF2 variants. In this report, we shed light on a novel missense mutation associated with cirrhosis in a family of brothers associated with phosphoinositide-3-kinase adapter protein 1 gene through high-output whole exosome gene sequencing methodology.

Aim: To compare the vertical root fractures (VRFs) resistance of teeth instrumented with ProTaper Universal, ProTaper Next, HyFlex CM and HERO Shaper nickel titanium (NiTi) rotary systems and obturated with compatible gutta-percha cones using the single-cone technique. Materials and Methods: The present study was performed in 72 extracted mandibular premolars. After removing the coronal parts of the teeth and determining working length, the roots were mounted in Eppendorf tubes. The samples were divided into five experimental groups, and one control group (n = 12). In Group 1, teeth were instrumented by hand K files; Group 2 ProTaper Universal (PTU); Group 3 HERO Shaper (HS); Group 4 HyFlex CM (HCM); Group 5 ProTaper Next (PTN); and Group 6 (Controls): the root canals were not shaped or filled. After the preparations were completed, roots were obturated with gutta percha. All the mounted samples were subjected to fracture resistance testing. Statistical Analysis: One-way ANOVA test and post hoc Tukey's test at a significance level of P < 0.05. Results: Fracture resistance of hand K file did not differ significantly from, HERO Shaper, HCM, and PTN NiTi rotary files (P = 0.929; P = 0.996; P = 1.000, respectively). PTU showed significantly less fracture resistance than hand K file (0.044). Among all NiTi rotary files, PTN depicted the highest fracture resistance, whereas PTU showed the least fracture resistance with statistically significant difference (P = 0.048). However, PTN did not differ significantly from HERO Shaper and HCM regarding fracture resistance. Conclusion: All rotary files showed similar fracture resistance values to Hand K file group except ProTaper Universal, which demonstrated significantly lower fracture resistance.

Atypical hemolytic uremic syndrome is a rare form of thrombotic microangiopathy caused by complement pathogenic variants. We describe a case of a 33-year-old woman who presented as rapidly progressing renal failure requiring dialysis and had anemia, microhematuria, low C3, normal C4 levels, and normal platelet count. Renal biopsy revealed arteriolar thrombotic microangiopathy and acute tubular injury. Patient was treated with plasma exchange and hemodialysis as required. This resulted in partial recovery at 1 month. Genetic workup by multiplex ligation-dependent probe amplification revealed a 1.5 times higher signal intensity on downstream region of CFH gene and 50% reduced intensity of exon 6 of CFHR1 gene, suggesting a gene conversion event, similar to those previously reported from Spain and Portugal.

To assess the knowledge of the general population and the medical specialists about the purpose and utility of cord blood banking. One hundred individuals from the general population and 100 clinicians from various departments were enrolled in the study between August 2013 and November 2013. Fifty eight percent of the doctors and 82 % of the lay persons did not know any indication or were not aware of the correct indications of the use of cord blood for transplantation. Around half of the lay persons (42 %) and doctors (37 %) thought that umbilical cord blood can be used to treat any genetic disorder including Duchenne muscular dystrophy and mental retardation. Ninteen percent of the doctors thought that umbilical cord blood can be used to treat thalassemia in the same child. The propaganda done by cord blood banks that cord blood is a biological insurance for the child is misleading and should be discouraged. The obstetricians and the pediatricians should take a central role in providing the correct information to would be parents to help them in taking a correct decision.

To create awareness about newborn screening (NBS) in underprivileged population and establish the model of NBS as an outreach program in peripheral hospitals in and around Lucknow.During this project, social workers were trained in taking informed consent, demographic details of newborns, heel prick samples and transporting the filter papers to a central laboratory. These social workers were posted in seven hospitals, in and around Lucknow, catering to families with low socioeconomic strata. Assays were performed in a single laboratory and results were conveyed back to the social workers and the hospitals.A total of 13,426 newborns were screened for the three conditions namely congenital hypothyroidism (CH), biotinidase deficiency and galactosemia. Over all coverage rate was 73.5 % and average time of reporting of results was 8.8 + 2.4 days. More than 85 % (86.7 %) families with positive screening test could be contacted back and out of them, 83.6 % babies could be sampled for confirmatory tests. Eleven babies were diagnosed to have CH. The study has shown positive and enthusiastic responses of the lay persons. Questionnaire based survey among lay persons showed that almost 100 % individuals understood the advantages and method of NBS.This project has been successful in establishing a model of NBS as an outreach program in and around a district.

Mucopolysaccharidosis IV A (Morquio syndrome A, MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS). The mutation spectrum in this condition is yet to be determined in Indians. We aimed to analyze the mutations in the GALNS gene in Asian Indians with MPS IVA. All the exons and the adjacent intronic regions of the gene were amplified and sequenced in sixty-eight unrelated Indian families. We identified 136 mutant alleles comprising of 40 different mutations. We report twenty-two novel mutations that comprise of seventeen missense (p.Asn32Thr, p.Leu36Arg, p.Pro52Leu, p.Pro77Ser, p.Cys79Arg, p.His142Pro, p.Tyr191Asp, p.Asn204Thr, p.Gly188Ser, p.Phe216Ser, p.Trp230Cys, p.Ala291Ser, p.Gly317Arg, p.His329Pro, p.Arg386Ser, p.Glu450Gly, p.Cys501Ser), three splice-site variants (c.120+1G>C, c.1003-3C>G, c.1139+1G>A), one nonsense mutation (p.Gln414*) and one frameshift mutation (p.Pro420Leufs*440). Eighteen mutations have been reported earlier. Among these p.Ser287Leu (8.82%), p.Phe216Ser (7.35%), p.Asn32Thr (6.61%) and p.Ala291Ser (5.88%) were the most frequent mutations in Indian patients but were rare in the mutational profiles reported in other populations. These results indicate that the Indian patients may have a distinct mutation spectrum compared to those of other populations. Mutant alleles in exon 1, 7 and 8 accounted for 44.8% of the mutations, and sequencing of these exons initially may be a cost-effective approach in Asian Indian patients. This is the largest study on molecular analysis of patients with MPS IVA reported in the literature, and the first report from India. (c) 2014 Wiley Periodicals, Inc.

Background and Introduction: Chromosomal abnormality is found in about half of first-trimester abortions. Karyotype is the gold standard to detect chromosomal abnormalities. Multiplex ligation-dependent probe amplification (MLPA) offers advantage over karyotype in terms of lower failure rate, faster turnaround time, and much higher resolution than conventional karyotyping and found to be 98% concordant with conventional karyotype. Aim: We performed this study to look for the utility of MLPA in diagnosing chromosomal abnormalities in first-trimester abortions. Materials and Methods: MLPA using subtelomeric SALSA probe sets (P036 and P070) was used to detect cytogenetic abnormalities in products of conception in missed/spontaneous abortions. Results: A total of ninety abortus samples were analyzed by MLPA. Successful results were provided in (67) 74.4% of the cases while no conclusion could be drawn in 25.6% (23) of the cases. Fifty-five (82.1%) cases were cytogenetically normal and 17.9% (12) had some abnormality. Aneuploidy was detected in 8 (66.7%) cases, 3 (25%) had double-segment imbalance, and one (8.3%) had partial aneuploidy. Conclusion: We suggest that MLPA is a good substitute to traditional karyotype.

Summary Background Hypohidrotic/anhidrotic ectodermal dysplasia (HED) is a rare Mendelian disorder affecting ectodermal tissues. The disease is primarily caused by inactivation of any one of three genes, namely ectodysplasin A1 (EDA-A1), which encodes a ligand belonging to the tumour necrosis factor (TNF) superfamily; ectodysplasin A receptor (EDAR), encoding the EDA-A1 receptor and ectodysplasin A receptor-associated death domain (EDARADD), encoding an adaptor protein. X-linked recessive (EDA-A1), the predominant form of HED, as well as autosomal recessive and dominant (EDAR and EDARADD) inheritance patterns have been identified in affected families. Objectives To determine the common genes causing HED in India. Methods We performed mutation analysis on 26 HED families from India (including 30 patients). In addition, we carried out sequence and structural analysis of missense/nonsense and insertion/deletion mutations. Results Among the 26 families analysed, disease-causing EDAR mutations were identified in 12 (46%) while EDA-A1 mutations were detected in 11 (42%). Four novel mutations in EDAR and five in EDA-A1 were identified. More importantly, a possible founder EDAR mutation, namely c.1144G>A, was identified in five independent families, thus accounting for about one-fifth of affected families in whom mutation was detected. A majority of EDA-A1 mutations localized to the TNF-like domain while the location of EDAR mutations was more widespread. Conclusions This is the first report of a founder EDAR mutation and of a significantly high frequency of autosomal recessive HED.

Objective The objective of this article was to ascertain the opinion of lay persons and medical practitioners in India regarding late termination of pregnancies (LTOP) for fetal abnormalities. Methods One hundred and fifty lay persons and 120 medical practitioners were given separate questionnaires and asked their opinions regarding LTOP for prenatally detected fetal abnormalities of varying severity. The views regarding legalisation of LTOP and the acceptability of feticide by the lay persons were also ascertained. Results More than two-thirds of the lay persons and majority (85.8%) of clinicians felt that LTOP should be allowed for fetal conditions with poor prognosis. At least 70% of lay persons felt that LTOP should be legalised for severe fetal abnormalities. For potentially treatable conditions, continuation of pregnancy in late gestation was the preferred option. For lethal malformations like anencephaly and disorders requiring lifelong treatment like meningomylocele and thalassemia major, majority of clinicians (86.7%, 69.2% and 55.8%, respectively) and lay persons (65%, 51% and 25%, respectively) had the opinion that termination of pregnancy can be offered at any gestational age. Conclusion Both the lay persons as well as the medical fraternity in India feel the need to look into revision of legalisation of LTOP particularly for fetal conditions with poor outcome. Copyright © 2011 John Wiley & Sons, Ltd.

Citation Agarwal M, Parveen F, Faridi RM, Phadke SR, Das V, Agrawal S. Recurrent pregnancy loss and apolipoprotein E gene polymorphisms: a case–control study from North India. Am J Reprod Immunol 2010; 64: 172–178 Problem The role of apolipoprotein E gene polymorphisms in the etiology of recurrent pregnancy loss (RPL) is not clearly understood. We evaluated this polymorphism in unexplained pregnancy losses among North Indian women. Method of study In a retrospective case–control study, 200 well-characterized RPL cases were examined for their APO-E genotypes based on restriction fragment length polymorphism analysis of PCR-amplified fragments including amino acid positions 112 and 158. The observed genotypes were compared with those obtained from an equal number of ethnically matched negative controls. Results We found similar APO-E genotypes and E2, E3, and E4 allele frequency distribution among RPL patients and controls. The allele frequencies obtained in patients and controls respectively were as follows: E2 = 7.5% and 9.0% (P = 0.52; OR = 0.81; 95%CI = 0.49–1.35), E3 = 89.7% and 90% (P = 1.00; OR = 0.97; 95%CI = 0.61–1.54), and E4 = 2.8% and 1% (P = 0.12; OR = 2.79; 95%CI = 0.88–8.86). Conclusions Our data did not support the association of APO-E gene polymorphisms with recurrent pregnancy loss as reported by some of the previous studies. We endorse adequate characterization of RPL cases, inclusion of appropriate negative controls, and adequate sample size prior to addressing such studies.

​Multicentric osteolysis nodulosis and arthropathy (MONA) is an infrequently described autosomal recessive skeletal dysplasia characterized by progressive osteolysis and arthropathy. Inactivating mutations in MMP2, encoding matrix metalloproteinase-2, are known to cause this disorder. Fifteen families with mutations in MMP2 have been reported in literature. In this study we screened thirteen individuals from eleven families for MMP2 mutations and identified eight mutations (five novel and three known variants). We characterize the clinical, radiographic and molecular findings in all individuals with molecularly proven MONA from the present cohort and previous reports, and provide a comprehensive review of the MMP2 related disorders.

Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are caused by loss of function of imprinted genes in the 15q11–13 critical region. Reports of PWS and AS in close relatives within the same family are rare. We report on the diagnosis of a familial unbalanced 10;15 translocation causing AS in a child that led to the prenatal diagnosis of an unbalanced 10;15 translocation with resultant deletion of the Prader–Willi critical region in her maternal uncle's offspring. © 2011 Wiley Periodicals, Inc.

To assess feasibility and recall rates for newborn screening for congenital hypothyroidism, galactosemia and biotinidase deficiency in a predominantly rural and inner city population in and around the City of Lucknow in Uttar Pradesh, India. Prospective observational study. Two tertiary-care and 5 district hospitals in and around Lucknow. All babies born in above hospitals during the study period. Heel prick samples were collected after 24 hours of life. Dried blood spot TSH, total galactose and biotinidase were assayed by immunofluorometry. Age related cut-offs were applied for recall for TSH. For galactosemia and biotinidase deficiency, manufacturer-suggested recall cut-offs used initially were modified after analysis of initial data. Recall rate for hypothyroidism, galactosemia and biotinidase deficiency. Screening was carried out for 13426 newborns, 73% of all deliveries. Eighty-five percent of those recalled for confirmatory sampling responded. Using fixed TSH cut off of 20 mIU/L yielded high recall rate of 1.39%, which decreased to 0.84% with use of age-related cut-offs. Mean TSH was higher in males, and in low birth weight and vaginally delivered babies. Eleven babies had congenital hypothyroidism. Recall rates with modified cut-offs for galactosemia and biotinidase deficiency were 0.32% and 0.16%, respectively. An outreach program for newborn screening can be successfully carried out in similar socio-cultural settings in India. For hypothyroidism, the high recall rate due to early discharge was addressed by age-related cut-offs.

We have developed a highly targeted custom next generation sequencing-based test targeting JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10, which are implicated in Philadelphia-negative chronic myeloproliferative neoplasms. The assay is capable of ultra-high sequencing depth and producing mutational detection sensitivities of 0.5% and below. We have validated the performance of the test through orthogonal testing and demonstrated a high degree of multiplexing with up to 50 samples in a single run. We show the clinical utility of this test through the description of a couple of cases with myeloproliferative disorders with Type-II and Type-II-like mutations in exon 9 of the CALR gene.

The objective of this article was to ascertain the opinion of lay persons and medical practitioners in India regarding late termination of pregnancies (LTOP) for fetal abnormalities.One hundred and fifty lay persons and 120 medical practitioners were given separate questionnaires and asked their opinions regarding LTOP for prenatally detected fetal abnormalities of varying severity. The views regarding legalisation of LTOP and the acceptability of feticide by the lay persons were also ascertained.More than two-thirds of the lay persons and majority (85.8%) of clinicians felt that LTOP should be allowed for fetal conditions with poor prognosis. At least 70% of lay persons felt that LTOP should be legalised for severe fetal abnormalities. For potentially treatable conditions, continuation of pregnancy in late gestation was the preferred option. For lethal malformations like anencephaly and disorders requiring lifelong treatment like meningomylocele and thalassemia major, majority of clinicians (86.7%, 69.2% and 55.8%, respectively) and lay persons (65%, 51% and 25%, respectively) had the opinion that termination of pregnancy can be offered at any gestational age.Both the lay persons as well as the medical fraternity in India feel the need to look into revision of legalisation of LTOP particularly for fetal conditions with poor outcome.